Biological validation of faecal corticosterone metabolites as a non-invasive stress assessment in translocated California valley quail (Callipepla californica)

Abstract US quail species are vulnerable to population declines as a result of climate change, habitat loss and habitat fragmentation, all of which can result in physiological stress. Additionally, population restoration techniques (PRTs), like translocations, also induce stress. Traditional assessments of avian stress hormone levels include capturing and handling birds to extract blood, methods that are inherently stressful and can compound stress analyses. However, the stress hormone corticosterone (CORT) is metabolized from the blood and excreted in faeces as faecal corticosterone metabolites (FCMs). FCMs have been used as a non-invasive measurement of stress hormone levels in a variety of species, but must be validated for each species. The objective of this study was to biologically validate the use of FCMs as a non-invasive measurement of CORT levels in California valley quail (Callipepla californica). Reference and treatment quail were acclimated for 3 weeks in an outdoor aviary. Subsequently, treatment quail were subjected to a simulated 48-h translocation, a common and stress hormone-inducing PRT. Faecal samples were collected every 4 h and processed using an enzyme immunoassay. Mean FCM concentrations of treatment quail (41.50 ± 16.13 ng/g) were higher than reference FCM concentrations (24.07 ± 10.4 ng/g). These results biologically validate the use of FCMs as a non-invasive method to assess CORT levels in California valley quail, demonstrate diurnal variation in quail CORT levels, and confirm that quail translocations are a stress-inducing PRT. Ultimately, this research validates a new non-invasive tool for stress response measurement to advance quail research, management and conservation.


Introduction
Stress assessments are often overlooked in US quail population declines and population restoration.US quail populations are vulnerable to decline as a result of unpredictable stressors like extreme weather, climate change (Guthery et al., 2000;Reyna and Burggren, 2017;Tanner et al., 2017;Reyna, 2019;Wilsey et al., 2019;Whitt and Reyna, 2022), habitat loss and habitat fragmentation (Brennan and Covington, 1994;Hernández et al., 2013;Pope and Heekin, 2017), all of which induce physiological stress responses.For example, elevated stress hormone levels have been observed in birds as a result of anthropogenic disturbances (Thiel et al., 2011;Jankowski et al., 2014;Arlettaz et al., 2015) and extreme climatic events (Wingfield et al., 2017), which are expected to increase in frequency and severity due to global climate change (Seneviratne et al., 2012;Masson-Delmotte et al., 2021).Climate change can also exacerbate the effects of other stressors (Felton et al., 2009;Şekercio glu et al., 2012).
Quail respond to stressors using two pathways: (1) a rapid, short-term response, mediated by the autonomic nervous system, which involves a rapid release of catecholamine from the adrenal medulla, and (2) a slower, long-term response, mediated by the hypothalamic-pituitary-adrenal (HPA) axis, which involves the release of corticosterone (CORT, 17deoxycortisol) from the adrenal cortex (Creel, 2001;Suorsa et al., 2003;Tempel and Gutiérrez, 2004;Cockrem, 2007Cockrem, , 2013;;Sheriff et al., 2011;Wingfield et al., 2017;Palme, 2019;Romero and Gormally, 2019;van Vliet et al., 2020).As a stress response, elevated CORT levels act as a catalyst that transitions quail into survival mode whilst suppressing nonessential functions.However, long-term elevated CORT levels disrupt normal bodily functions and can lead to disease and death (Blas and Fairhurst, 2022).Accordingly, CORT's role in the stress response, and the detectability of its metabolites using non-invasive methods, make it a common proxy for assessing the magnitude of an animal's stress response (Sheriff et al., 2010;Ellis et al., 2012).
Prolonged or repeated exposure to stressors can directly influence bird populations by altering foraging, predator avoidance, fledging and reproduction, (Cyr and Romero, 2007;Crespi et al., 2013;Martin et al., 2017;Wingfield et al., 2017).Stressor persistence can induce a type 1 allostatic overload, where energy demand exceeds energy availability (Blas and Fairhurst, 2022), and birds decrease or eliminate behaviours (e.g.reproduction) not immediately necessary for survival (McEwen and Wingfield, 2003).Reproduction may be interrupted through several different, but complimentary pathways, including increasing production of gonadotropininhibitory hormone, reducing production of gonadotropinreleasing hormone (GnRH), and decreasing sensitivity of pituitary gonadotrope cells to GnRH and of ovaries to luteinizing hormone (Sapolsky et al., 2000;Wingfield and Sapolsky, 2003;Dickens and Bentley, 2014;Son et al., 2014).For example, capturing and holding wild European starlings (Sturnus vulgaris) blocked a time-sensitive seasonal increase in HPA axis activity, preventing the transition from early gonadal development to an active breeding state, which inhibited breeding (Dickens and Bentley, 2014).The resultant reduction in reproduction in birds contributes to population declines.Chronic stress can also disrupt acute stress responses, including the fight or flight response, due to a decrease in the sensitivity of the HPA axis (Rich and Romero, 2005;Romero and Butler, 2007;Dickens et al., 2009).For example, European starlings caught in the wild and placed into captivity lost the ability to produce a fight or flight response when exposed to a loud noise as a stressor (Dickens and Romero, 2009).In addition, when the fight or flight response was chronically stimulated, it negatively impacted cardiovascular health, leading to hypertension due to surplus catecholamine exposure (Rupp, 1999).Population restoration techniques (PRTs), like translocations, are also stressors that can increase CORT levels.US quail species are economically important gamebirds (Johnson et al., 2012;Wszola et al., 2020) and part of a $3.7 billion per year upland gamebird hunting industry (Sportsmen's Alliance, 2021).For >150 years, translocations have been used in attempts to bolster populations (Hernández et al., 2013;Gomez and Reyna, 2017;Whitt et al., 2017).Quail translocations are rarely successful (Perez et al., 2002;Scott et al., 2013;Whitt et al., 2017), especially when used for reintroduction (Martin et al., 2017).Despite translocations rarely having long-term success, there is increasing interest in translocation of wild quail (Stephenson et al., 2011;Martin et al., 2017;Sisson et al., 2017).Numerous hypotheses have been proposed for the low success rate (Sokos et al., 2008;Martin et al., 2017), but little is known about how stress influences translocation success.
Translocation consists of capturing, handling, holding, transporting and releasing quail to a novel site, each of which can result in acute and chronic stress, independent of other processes (Buchanan, 2000;Fazio and Ferlazzo, 2003;Jones et al., 2005;Teixeira et al., 2007;Dickens et al., 2009Dickens et al., , 2010;;Wingfield and Romero, 2011;Batson et al., 2017;Martin et al., 2017).For example, short translocations may induce an acute stress response; however, the duration of a quail translocation is typically 1-3 days, which increases the occurrence of a chronic stress response as indicated by chronically elevated CORT levels (Dickens et al., 2010).Both chronic and acute stress responses can inhibit survival of translocated animals through increased energy requirements, capture myopathy, dispersal distance and weight loss (Armstrong and Seddon, 2008;Dickens et al., 2010;Breed et al., 2019), which lead to a failure of the translocation by increasing susceptibility to disease and death (Dickens et al., 2010;Martin et al., 2017).
Weight loss has been observed when translocating wild birds to novel environments (Rich and Romero, 2005;Dickens et al., 2009;Fischer et al., 2018), and is a response to chronic stress.For example, European starlings lost 5-15% of their body weight after being exposed to a 14-day regime of multiple stressors during a translocation (Awerman and Romero, 2010).California valley quail (C.californica) exhibited a mean weight loss of 14.3% when translocated from Idaho to Texas (Reyna et al., 2020).This is important because body mass can influence survival and the overall success of translocations.For example, Cirl buntings (Emberiza cirlus) with a higher body weight at capture were more likely to survive in their new habitat than those with lower body weights (Fountain et al., 2017).Reducing stressors during translocation could reduce weight loss and increase postrelease survival (Warwick et al., 2006).
The traditional method of measuring stress in birds requires capturing, handling and extracting blood samples, inherently stress-inducing actions (Arnold et al., 2008), followed by measuring blood CORT levels.It is assumed that if this process occurs within 3 min of initial handling, CORT levels will be indicative of the physiological condition of the bird prior and will not represent the elevated stress incurred during the procedure (Littin and Cockrem, 2001;Cockrem and Silverin, 2002).However, CORT levels quickly rise during handling and peak within 15-30 min.In addition, handling associated with blood extractions adds to the cumulative stress experienced by the bird, and nearby birds, independent of the extraction (Dickens et al., 2010;O'Dell et al., 2014;Wein et al., 2017).
Steroid hormones, like CORT, can also be measured from urine, faeces, hair and feather samples (Bortolotti et al., 2008;Sheriff et al., 2011).One effective, non-invasive and increasingly popular method to evaluate a stress response is by measuring faecal corticosterone metabolites (FCMs; Romero and Remage-Healey, 2000;Tempel and Gutiérrez, 2004;Möstl et al., 2005;Dickens et al., 2009;Sheriff et al., 2010).Measuring FCMs as an assessment of CORT levels has been successful in a wide range of avian studies (Washburn et al., 2003;Fletcher et al., 2018;Sokół and Koziatek-Sadłowska, 2020).Excretion of FCMs varies between species, as does the suitability of different immunoassays to accurately detect CORT; therefore methods must be validated for each species (Palme et al., 1996;Wasser et al., 2000;Palme, 2019).Physiological validation of measuring FCM concentrations requires injecting a known quantity of CORT into an animal (Touma and Palme, 2005), an invasive and stressful procedure (Palme, 2019;Mohlman et al., 2020).Biological validation of measuring FCM concentrations is a noninvasive procedure where faeces is collected before and after an event that increases HPA activity for an extended time, resulting in increased blood CORT levels (Touma and Palme, 2005;Palme, 2019).
The goal of this study was to biologically validate the use of FCMs as a non-invasive assessment of CORT levels in California valley quail using a simulated translocation as the stressor.This is the first biological validation of FCMs as a tool to detect elevated CORT levels in a new world quail species.Mohlman et al. (2020) performed a physiological validation of FCMs with northern bobwhite (Colinus virginianus), another new world quail, but were unsuccessful at a biological validation due to an ineffective biological stressor.

Materials and Methods
Flight-ready captive-reared valley quail (n = 63 females, n = 57 males), 16-24 weeks in age, were acquired from a breeder (ZKD Game Birds, West Point, TX, USA) in October, 2020.Thirteen valley quail (n = 5 females, n = 7 males) were selected at random to use for biological validation, with the remainder used for diurnal FCM analysis.Quail were housed and acclimated in groups in an outdoor aviary (Quail Hotel, Fannin Fabrication, Bonham, TX, USA), where they were provided with natural perches and dust baths for 3 weeks (Hawkins et al., 2001).Mean temperature during this period was 16.4 • C. Birds were provided with gamebird feed (Gamebird Starter and Grower, M-G Inc., Weimar, TX, USA) and water ad libitum.This mimicked the time and conditions in which quail were held prior to the first shipment of trapped birds during a 2019 translocation of valley quail (Reyna et al., 2020).Acclimation was assumed when birds exhibited typical behaviour (e.g.eating, drinking, roosting, grooming; Reyna and Newman, 2018).
To biologically validate FCMs in valley quail, reference (low stress) faecal samples were obtained from acclimated valley quail.The floor of the aviary was lined with a plastic sheet.The birds were observed from ∼50 m away with binoculars to record the sex of the bird, and to ensure collected faecal samples were fresh (<1 h old) and uncontaminated.Once collected, individual faecal samples were placed into a plastic storage bag (Ziploc freezer quart, S. C. Johnson & Son, Inc., Racine, WI) and labelled with the date, time and sex of the bird.Because individual birds could not be matched to collected faecal samples, reference sample data were pooled following FCM analysis.Ambient temperature, relative humidity and presence of direct sunlight were recorded on the plastic storage bag, since these factors can degrade hormone detection probability (Shipley et al., 2019).Immediately after collection and labelling, samples were stored in a freezer at −20 • C until FCM analysis (Wasser et al., 1988;Messmann et al., 1999;Khan et al., 2002).
For biological validation, quail were transported to the animal care facility at Texas A&M University-Commerce to undergo a simulated 48-h translocation, a stressful event (Martin et al., 2017).Each quail was given a unique leg band and had their age, sex, and weight recorded.collected from the underlying waste pan trays 4-5 times daily (Millspaugh and Washburn, 2004;Mohlman et al., 2020).The frequency of faeces collection ensured a representative sample, since FCMs may fluctuate diurnally (Breuner et al., 1999).Individual faecal samples were placed in a plastic storage bag and labelled with the bird ID number, sex, time, and date of collection.Steroid hormone concentrations can be affected by storage duration (Millspaugh and Washburn, 2004) and bacteria (Lexen et al., 2008).Therefore, treatment faecal samples were immediately stored in a freezer (−20 • C) until FCM analysis (Wasser et al., 1988;Messmann et al., 1999).
FCM concentrations of reference and treatment quail faecal samples were measured using an Enzyme Immunoassay (EIA) Kit (K-014H, Arbor Assays, Ann Arbor, MI, USA).The kit was validated for use on CORT extracted from dry faecal extracts on a multi-species design (DeVries and Jawor, 2013;Fletcher et al., 2018), and included detailed instructions for extracting CORT from faecal samples and analysing FCM concentrations.To determine the assays suitability for valley quail, 10 faecal extracts were pooled from the valley quail simulated translocation, and a serial dilution (n = 6 dilutions) was performed.FCM concentrations from the pooled faecal extracts were parallel to the slope of the standard curve, verifying suitability for valley quail.The sensitivity of the EIA was determined to be 18.19 pg/ml.Optical density was calculated using a Synergy LX Multi-mode microplate reader (Bio Tek Instruments, USA), and entered into MyAssay web (https://www.myassay.com),to calculate FCM concentration in picograms per millilitre.FCM concentrations reported in picograms per millilitre were converted to nanogramme/gramme for comparison to other studies.
All animals were handled in accordance with procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2010), and Texas A&M University-Commerce Animal Use Protocol P20-013.

Statistical analysis
Because birds could not reliably be distinguished without technicians approaching close enough to elicit visible agitation and warning calls from the reference birds, we pooled samples for statistical comparison.All statistical analyses were conducted in R (version 4.0.2,R Foundation, Austria).Shapiro-Wilk's statistical test was used to check for normality (Zar, 1996).Welch's 2-sample t-test was used to calculate the P-value, mean and 95% confidence interval between 2 variables with normal distributions.Mann-Whitney U-test was used to compare 2 variables with non-normal distributions.Kruskal-Wallis test was used to compare multiple variables with non-normal distributions, followed by Dunn's test with Bonferroni correction for pairwise comparisons.Correlations were tested using Pearson's product-moment correlation with Bonferroni correction (Zar, 1996).All weight data are presented as mean ± standard deviation (SD).Results were significant at alpha < 0.05.
Valley quail with higher initial body mass prior to the simulated translocation experienced lower mean FCM concentrations (r 2 = 0.14, P < 0.001), and lower percentage of initial mass lost (r 2 = 0.13, P < 0.001) during the simulated translocation.FCM concentrations positively correlated with percent of initial body mass loss (r 2 = 0.27, P < 0.001).No difference in FCM concentrations (Mann-Whitney U, Z = 0.15, P = 0.44), total mass lost (Welch's t-test, P = 0.58) or percentage of initial mass lost (Welch's t-test, P = 0.29) was observed based on sex.

Discussion
This study was the first biological validation of FCMs as a non-invasive assessment of a stress response for a new world quail species.When exposed to a simulated translocation, valley quail experienced a 72.4% increase in FCMs  compared to reference concentrations, indicating that this protocol successfully detected an increase in FCMs during a stressful event.These results are comparable to the 73% increase in FCMs recorded in northern bobwhites, another new world quail, during a physiological validation (Mohlman et al., 2020).However, the increase in FCM levels recorded for both of these new world quail is lower than FCM levels recorded in other avian species.This is not surprising because excretions of metabolites can vary between species (Palme et al., 1996;Wasser et al., 2000;Palme, 2019).For example, experimentally stressed European starlings showed a ∼100% increase in FCMs (Cyr and Romero, 2008).Following capture and holding for a veterinary examination, African penguins (Spheniscus demersus) showed a 155-349% increase in FCMs.Wild Dickcissels (Spiza americana) experienced a 1700% increase in FCM concentrations within 24 h of having leg harness transmitters attached (Suedkamp Wells et al., 2003).Valley quail and northern bobwhite quail are both new world quail, and their similar stress response during validation, and variation from other taxa, was expected.This emphasizes the need to validate FCM for individual species.
The correlation of weight loss with FCM levels further supports the association of chronic stress with weight loss in birds (Cyr and Romero, 2007;Dickens et al., 2009;Awerman and Romero, 2010).Quail in our study lost 11.6% of body weight during the 48-h simulated translocation, results consistent with weight loss in translocated starlings (5-15%) and wild valley quail (14.3%) in an actual translocation of similar duration (Reyna et al., 2020), indicating that translocation weight loss may be a result of a chronic stress response to handling and captivity.Similar to our study, Reyna et al. (2020) reported that heavier valley quail lost less weight than lighter valley quail during a translocation.Heavier birds may be healthier overall, and future studies may benefit from considering a minimum weight for translocation.
By collecting faecal samples 4-5 times daily during the simulated translocation, we were able to capture diurnal variation in FCM concentrations.Although not previously observed in new world quail (Mohlman et al., 2020), diurnal variation in CORT metabolites has been recorded in a variety of species (Florant and Weitzman, 1980;de Jong et al., 2001;Touma et al., 2004;Bosson et al., 2009;Sheriff et al., 2009), generally corresponding to the circadian rhythm of the animal.The diurnal variation in valley quail FCM concentrations followed a pattern similar to the variation in plasma CORT observed in broiler chickens (de Jong et al., 2001), with lower concentrations at night and elevated concentrations during the day.This trend was expected since valley quail are active in the day and inactive during the night (Leopold, 1977).
This study verifies that translocations are stressful events for quail that result in significant weight loss, results that suggest stress influences translocation success.Chronic stress is associated with increased mortality in starlings (Cyr and Romero, 2007;Dickens et al., 2010), and animal weight at release is a predictor of survival in cirl buntings (Fountain et al., 2017).Further, increased CORT levels can inhibit reproduction, a primary indicator of translocation success (Griffith et al., 1989;Dickens et al., 2010).Future studies could benefit by focusing efforts on stress mitigation to increase long-term translocation success.

Figure 2 :
Figure 2: Diurnal variation in FCM concentrations from California valley quail during a simulated 48-h translocation.Treatment FCM concentrations correlated with photoperiod (12 L:12 D) and quail circadian rhythm.Letters indicate statistical groupings.Faecal sample numbers ranged from 9 to 37 per hour due to faecal dropping frequency.